Cytochrome P450 transcriptional enhancer nucleic acid molecule

ABSTRACT

An isolated nucleic acid molecule comprising a nucleotide sequence encoding a transcriptional enhancer of cytochrome P450 (P450) CYP3A4 production or expression is disclosed, as are uses of the nucleic acid molecule for screening compounds for xenobiotic induction of CYP3A4 expression in cells and animals.

TECHNICAL FIELD

The present invention relates to modulating or effecting gene expressionand/or formation of human and non-human animal cytochrome P450 CYP3Asubfamily enzymes.

BACKGROUND ART

Development of therapeutic drugs for medical and veterinary use isimportant in order to improve and advance treatment modalities in humansand animals. Unfortunately, many drugs that are developed andsubsequently used have been found to have limited half lives in vivo dueto rapid metabolism by the recipient. It would be advantageous to have asystem for screening potential new drugs for susceptibility to metabolicaction in patients.

A cytochrome P450 (P450) termed CYP3A4 is an important human gene thatcodes for an enzyme expressed in the liver, and to a lesser extent arange of other tissues. It is quantitatively the most abundant P450 inhuman liver. The CYP3A4 enzyme is pivotal to the metabolism of manyexogenous chemicals (xenobiotics), including therapeutic drugs, as wellas a range of endogenous compounds, such as steroid hormones. Changes inthe level of expression of CYP3A4 can dramatically affect the rate ofelimination of therapeutic drugs and thus impact on their effectiveness.A range of exogenous chemicals, including some therapeutic drugs,(hereafter termed ‘xenobiotic inducers’) are able to increase thetranscriptional rate of the CYP3A4 gene and hence the formation of theCYP3A4 enzyme. The result is the elimination of drugs metabolised byCYP3A4 being greatly increased thereby diminishing their therapeuticeffect.

The present inventors have obtained a DNA molecule, lying approximately7.5 kilobases 5′ to the transcription initiation site of the human P450CYP3A4 gene, that is responsible for the transcriptional induction ofthe CYP3A4 gene by xenobiotic inducers, including therapeutic drugs andis also involved in the constitutive expression of this gene. Thisnucleic acid molecule, called a ‘xenobiotic-responsive enhancer module’(XREM) by the present inventors, has a number of beneficial uses.

DISCLOSURE OF INVENTION

In a first aspect, the present invention consists in an isolated nucleicacid molecule including a nucleotide sequence forming a transcriptionalenhancer of cytochrome P450 (P450) CYP3A4 production or expression.

Preferably, the isolated nucleic acid molecule includes a nucleotidesequence substantially as shown in FIG. 1 (SEQ ID NO: 1), or afunctionally equivalent nucleotide sequence or portion thereof encodingan enhancer of CYP3A4, or a sequence which hybridises to the nucleotidesequence of FIG. 1 (SEQ ID NO: 1), or a sequence which shows at least60% homology with the nucleotide sequence of FIG. 1 (SEQ ID NO: 1). Morepreferably, the nucleic acid molecule has at least 80% homology with thenucleotide sequence of FIG. 1 (SEQ ID NO: 1) and most preferably thenucleic acid molecule has at least 90% homology with that sequence.

In a preferred embodiment, the present invention consists in an isolatednucleic acid molecule including nuclear receptor response elements fromthe 5′-flanking region of CYP3A4. Preferably, the response elements areselected from

-   XREM-DR3-1 GAA TGAACTTGC TGACCC TCT (SEQ ID NO: 2);-   XREM-ER6 CCT TGAAAT CATGTC GGTTCA AGC (SEQ ID NO: 3);-   XREM-DR6 AGG TGAATC ACAAGC TGAACT TCT (SEQ ID NO: 4);-   XREM-DR3-2 ATA TATTGT TAT TGAACT ATC (SEQ ID NO: 5); and-   Prox-ER6 ATA TGAACT CAAAGG AGGTCA GTG (SEQ ID NO: 6).

As a number of specific response elements have been identified in theenhancer of CYP3A4 by the present inventors, it will be appreciated thatSEQ ID NO: 1 can be used to identify other response elements. As thefull sequence of the enhancer is not necessary for subsequent use, thepresent invention includes within its scope the use of response elementsfrom the enhancer with intermediate or connecting sequences from othersources.

Preferably, the isolated nucleic acid molecule has a nucleotide sequencesubstantially as shown in FIG. 1 (SEQ ID NO: 1).

In a preferred form, the CYP3A4 is human CYP3A4. It will be appreciated,however, that the present invention also includes other human CYP3Asubfamily enzymes and CYP3A subfamily enzymes from non-human animals.

The induction of CYP3A4 is preferably by one or more xenobioticinducers.

The present invention also includes polynucleotides which hybridise tothe sequence shown in FIG. 1 (SEQ ID NO: 1). Preferably, thepolynucleotide hybridises to the sequence set out in FIG. 1 (SEQ IDNO: 1) under high stringency. As used herein, stringent conditions arethose that (a) employ low ionic strength and high temperature forwashing, for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1% NaDodSO₄at 50° C.; (b) employ during hybridisation a denaturing agent such asformamide, for example, 50% (vol/vol) form amide with 0.1% bovine serumalbumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphatebuffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42° C.; or(c) employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate),50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 g/ml), 0.1% SDS and10% dextran sulfate at 42° C. in 0.2×SSC and 0.1% SDS.

In a further preferred embodiment of the first aspect of the presentinvention, the polynucleotide capable of hybridising to the nucleotidesequence of FIG. 1 (SEQ ID NO: 1) is less than 5000 nucleotides,however, it can be less than 1000 or even less than 500 nucleotides inlength. Preferably, the hybridising polynucleotides are at least 10,more preferably at least 18 nucleotides in length.

In a second aspect, the present invention consists in a geneticconstruct suitable for use in measuring the induction of a reportergene, the construct including a nucleic acid molecule according to thefirst aspect of the present invention operably linked to a nucleic acidmolecule encoding a reporter molecule.

Preferably, the nucleic acid molecule encoding the reporter moleculeencodes an enzyme. The nucleic acid molecule encoding the reporter mayencode the enzyme CYP3A4 or a functionally equivalent enzyme. Examplesof suitable reporter molecules include firefly luciferase,beta-galactosidase, chloramphenicol acetyltransferase, alkalinephosphatase and green fluorescent protein. Alternatively, the nucleicacid may encode a messenger RNA (mRNA) that can be detected by standardtechniques such as northern blotting or ribonuclease protection assays.

It will be appreciated, however, that the nucleic acid molecule encodingthe reporter molecule may be any nucleic acid molecule or gene that,when expressed, has a detectable activity. The nucleic acid moleculeencoding reporter molecule does not have to be associated with thecytochrome P450 system although the inducer of transcription (XREM) usedis derived from that gene system. It will also be appreciated that thenucleic acid molecule encoding the reporter gene may include more thanone reporter molecule.

In a third aspect, the present invention consists in an assay systemincluding the construct according to the second aspect of the presentinvention such that on exposure of the system to a xenobiotic inducer,expression of the nucleic acid molecule encoding the reporter moleculeis enhanced.

The assay system includes within its scope cells containing theconstruct (XREM) and cell-free systems capable of supporting thefunction of the construct (XREM). Such cell-free systems typicallycontain cell extracts, such as nuclear extracts, but not always. Theassay system also includes experimental systems that determine bindingof compounds to the construct (XREM). These include DNase I footprintingand gel-retardation assays. These assay systems can be used to detectactivation of the construct (XREM), but are not as convenient for massscreening of compounds as the reporter gene constructs. Preferably, theenhanced expression results in an increase in the activity of thereporter gene product.

The cell may be any suitable cell including bacterial, plant or animalcells. The construct may exist as a separate genetic entity in the cellor be incorporated into the genome of the cell. Furthermore, the cellmay form part of a transgenic animal.

In a fourth aspect, the present invention consists in a method forscreening a compound for xenobiotic induction of CYP3A4 expression in acell, the method including exposing an assay system according to thethird aspect of the present invention to the compound and measuring forthe induction or the potential for expression of the nucleic acidmolecule encoding the reporter molecule.

The method according to the present invention is particularly suitablefor screening new therapeutic drugs. Although there may be manypotential drugs available at the developmental stage, if a drug inducesCYP3A4 expression in vivo then its suitability as an effectivetherapeutic agent is reduced. In use, the drug's half life will beshorter due to being metabolised by the induced CYP3A4 enzyme in theliver of a patient. Another problem that can arise is that the drugcauses the enhanced metabolism or elimination of other drugs given tothe patient. Drug “cocktails” or combinations of drugs are oftenrequired to treat many diseases. If one of the drugs administered hasthe propensity to enhance the clearance of one or more other drugs usedin the treatment of a particular disease, then this is highlyundesirable and may result in the reduced efficacy of the treatment.

It will be appreciated that if a compound or new drug fails to inducethe expression of the reporter gene when tested by the method accordingto the present invention, then this is an indication that the compoundor new drug may not be an xenobiotic inducer and therefore a suitablecandidate for further development. A screening process would bebeneficial in therapeutic drug development as unsuitable candidates maybe disregarded at an early of stage of development. Furthermore,alternate or related chemical compounds may also be developed based on acompound's negative result in the screening assay.

The method may also include exposing a transgenic animal and measuringin the animal for induction of the reporter gene after exposure to thechemical or drug.

In a fifth aspect, the present invention consists in the XREM accordingto the first aspect of the present invention as a genetic analysis tool.In this respect, mutations responding to allelic variants in humans areintroduced and their functional consequences observed. Also, as the XREMhas a defined function of transcriptional regulation it can be used forthe determination of allelic variation.

In a further aspect, the use involves the determination of allelicvariation within the XREM (relevant to constitutive expression) and theuse of site directed mutation of the XREM to determine the impact ofallelic variation.

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element, integeror step, or group of elements, integers or steps, but not the exclusionof any other element, integer or step, or group of elements, integers orsteps.

In order that the present invention may be more clearly understood,preferred forms will be described with reference to the followingexamples and drawings.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a sequence of the XREM region of the CYP3A4 gene (SEQ IDNO: 1). Base numbering is relative and does not indicate location withinthe CYP3A4 gene structure.

FIG. 2 shows an example of a series of deletion constructs of the5′-flanking region of the CYP3A4 gene used to definexenobiotic-responsive elements. Fold induction of luciferase reportergene expression refers to treatment with a potent CYP3A4 inducer (5 μMrifampicin) compared to vehicle alone (0.1% dimethyl sulphoxide). Thiswas normalised to the activity of an ‘empty’ pGL3-basic reporter vector.All constructs were temporarily transfected into HepG2 cells.

FIG. 3 shows the effect of cotransfection of a hPXR expression vector(pSG-hPxR) on HepG2 cells containing an CYP3A4 XREM reporter construct,in the presence or absence of a potent CYP3A4 inducer (5 μM rifampicin).The insert shows a magnified view of the effects on the control cells.

FIG. 4 shows the effect of various inducing drugs on HepG2 cellscontaining a CYP3A4 XREM reporter construct and a hPXR expression vectorcompared to vehicle (dimethyl sulphoxide) 0.1% alone. Legend: 1, DMSO;2, rifampicin; 3, RU-486; 4, clotrimazole; 5, phenobarbital; 6,metyrapone; 7, pregnenalone 16α-carbonitrile.

FIG. 5 shows the effect of deletions and site-directed mutagenesis ofputative nuclear receptor response elements on the transcriptionalinduction of the CYP3A4 5′-flanking region by 5 μM rifampicin. Filled inboxes correspond to mutated elements. Numbering is relative to thetranscription initiation site.

FIG. 6 shows an optimised CYP3A4 5′-flanking region construct coupled toa luciferase reporter gene (Luc) for determining the ability ofxenobiotics to induce transcriptional activation of the CYP3A4 gene.Numbering is relative to the transcription initiation site.

MODES FOR CARRYING OUT THE INVENTION

Definitions

General Molecular Biology

Unless otherwise indicated, the recombinant DNA techniques utilised inthe present invention are standard procedures, well known to thoseskilled in the art. Such techniques are described and explainedthroughout the literature in sources such as, J. Perbal, A PracticalGuide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook etal., Molecular Cloning: A Laboratory Manual, Cold Spring HarbourLaboratory Press (1989), T. A. Brown (editor), Essential MolecularBiology: A Practical Approach. Volumes 1 and 2, IRL Press (1991), D. M.Glover and B. D. Hames (editors), DNA Cloning: A Practical Approach,Volumes 1–4, IRL Press (1995 and 1996), and F. M. Ausubel et al.(Editors), Current Protocols in Molecular Biology, Greene Pub.Associates and Wiley-Interscience (1988, including all updates untilpresent) and are incorporated herein by reference.

Mutants, Variants and Homology—Nucleic Acids

Mutant polynucleotides will possess one or more mutations which aredeletions, insertions, or substitutions of nucleotide residues. Mutantscan be either naturally occurring (that is to say, isolated from anatural source) or synthetic (for example, by performing site-directedmutagensis on the DNA). It is thus apparent that polynucleotides of theinvention can be either naturally occurring or recombinant (that is tosay prepared using recombinant DNA techniques).

An allelic variant will be a variant that is naturally occurring withinan individual organism.

Nucleotide sequences are homologous if they are related by divergencefrom a common ancestor. Consequently, a species homologue of thepolynucleotide will be the equivalent polynucleotide which occursnaturally in another species. Within any one species a homologue mayexist as numerous allelic variants, and these will be consideredhomologues of the polynucleotide. Allelic variants and specieshomologues can be obtained by following standard techniques known tothose skilled in the art. Preferred species homologues include thoseobtained from representatives of the same Phylum, more preferably thesame Class and even more preferably the same Order.

A polynucleotide at least 70% identical, as determined by methods wellknown to those skilled in the art (for example, the method described bySmith, T. F. and Waterman, M. S. (1981) Ad. Appl. Math., 2: 482–489, orNeedleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol., 48: 443–453),to the that of the present invention are included in the invention, asare proteins at least 80% or 90% and more preferably at least 95%identical to the polynucleotide of the present invention. This willgenerally be over a region of at least 60, preferably at least 90,contiguous nucleotide residues.

Substantially Purified

By “substantially purified” the present inventors mean a polynucleotidethat has been separated from lipids, nucleic acids, other polypeptidesor polynucleotides, and other contaminating molecules.

Active Fragment

By “active fragment” the present inventors mean a fragment of a sequenceshown in FIG. 1 which encodes a transcriptional enhancer of cytochromeP450 (P450) termed CYP3A4.

BACKGROUND

Human cytochrome P450 3A4 (CYP3A4)

CYP3A4 is an important gene in human clinical pharmacology. In additionto it's pivotal role in the metabolism of therapeutic drugs andendogenous steroid hormones, recent studies have associated a non-codingpolymorphisms in a putative regulatory region of this gene with bothprostate cancer and the development of secondary leukaemia after cancerchemotherapy. An understanding of CYP3A4 gene regulation is important inthe development of therapeutic drugs. In addition, CYP3A4 generepresents a novel target for pharmacological manipulation and a toolfor both pharmacogenetic analysis and possibly disease prediction.However, a thorough understanding of CYP3A4 regulation and genetics isrequired to achieve these goals. This present invention is directed to aregulatory module within the 5′-flanking region of the CYP3A4 gene thatis responsible for it's xenobiotic induction and constitutiveexpression.

Human Hepatic Cytochromes P450s

The human hepatic P450s are membrane bound haemoproteins active in themetabolism of a broad range of lipophilic substrates. P450 proteins areof considerable relevance to medicine, not only because of their centralrole in drug and other xenobiotic metabolism, but also because of theirmany ‘natural’ or ‘endogenous’ lipophilic substrates. These includesteroid hormones, fatty acids (especially leukotrienes), cholesterol,and bile salts. It is considered likely that the P450 system evolvedbecause of its pivotal role in cholesterol and steroid metabolism. Asmall subset of constitutively expressed human hepatic P450s, includingCYP3A4, are of particular interest because they are quantitatively themost important forms and catalyse a range of biologically importantreactions.

Physiological Role and Variability of CYP3A4

CYP3A4, the predominant cytochrome P450 found in human liver, comprisesup to 60% of the total amount of hepatic cytochrome P450 protein. CYP3A4is involved in the metabolism of an extensive range of drugs andendogenous steroids. It has been estimated that in excess of 60% of alltherapeutic drugs are metabolised in full or in part by this enzyme.Changes in the level of expression of CYP3A4 can have a dramatic impacton the metabolism of therapeutic drugs and thus cause a number ofimportant drug interactions. Also, the variable level of constitutiveexpression of this enzyme makes a major contribution to theinter-individual variability of oxidative drug metabolism. In addition,CYP3A4 is the major pathway of oxidative metabolism of steroid hormones,catalysing the 6β-hydroxylation of several endogenous steroids such ascortisol, androstenedione and testosterone as well as the 2- and4-hydroxylation of estradiol to form catachol estrogens. Two other CYP3Asubfamily cytochromes P450 are found in man. CYP3A7 is mainly found infoetal liver while CYP3A5 is found mainly in adult liver as well as thelungs and kidneys.

Regulation of CYP3A4 Gene Expression

CYP3A4 is subject to multiple levels of transcriptional regulation.These include xenobiotic induction (for example, by some therapeuticdrugs), tissue-specific regulation, as well as substantial variabilityin constitutive expression (up to 10-fold between individuals notexposed to known inducers or inhibitors).

Xenobiotic Induction of CYP3A Genes

CYP3A genes in several mammalian species, including man, aretranscriptionally-inducible by a number of structurally dissimilartherapeutic drugs. Working on the assumption that xenobiotic inductionof CYP3A4 is mediated by a response element in the 5′-flanking region ofthis gene, the present inventors went on to clone 22 kilobases (kb) ofthe CYP3A4 gene 5′ to the transcription initiation site. This was usedto generate a deletion series of constructs covering 13 kb of the5′-flanking region that were linked to a luciferase reporter gene foranalysis of transcriptional activity. These constructs were temporarilytransfected into a human hepatoblastoma cell line (HepG2) as well asprimary cultures of rat and rabbit hepatocytes. By comparing transfectedcells treated with inducing agents, such as rifampicin, to untreatedcontrols, the present inventors have determined that the responseelement [termed hereafter a xenobiotic-responsive enhancer module(XREM)] is approximately located between −7.2 kb and −7.8 kb 5′ to thetranscription initiation site (FIG. 2). The present studies show theXREM to be a complex element spanning several hundred base pairs (FIG.1, SEQ ID NO: 1). DNase I footprinting further shows that this region isrich in DNA-protein interactions and appears capable of binding severaltranscription factors of which the recently described human pregnane-Xreceptor (hPXR) (the pregnane-X receptor is identical to thepregnane-activated receptor (PAR) and the steroid-X receptor (SXR))[1–3] is the most important for xenobiotic-induced regulation of CYP3A4.

The present inventors have demonstrated that the hPXR forms the vitallink between the xenobiotic inducer (eg, rifampicin) and elements withinthe XREM. In humans, rifampicin treatment typically induces theexpression of the CYP3A4 gene 50-fold or more. When a suitable reporterconstruct containing the XREM sequence is transfected into HepG2 cells,rifampicin treatment results in a 3- to 5-fold induction of reportergene expression. However, the present inventors have demonstrated thatco-transfection of the XREM-containing reporter gene construct with ahPXR expression vector restores full rifampicin induction (FIG. 3). Thepresent inventors have also shown that other drugs, such asphenobarbitone and clotrimazole also act on the XREM via the hPXR (FIG.4), demonstrating that this is a general mechanism for xenobioticinduction of the CYP3A4 gene.

The hPXR is an orphan nuclear receptor, belonging to the nuclearreceptor gene superfamily [4]. Studies from other laboratories suggestthat it binds to DNA as a heterodimer with the retinoid-X receptor (RXR)[1–3]. The present inventors have identified several putative nuclearreceptor response elements within the XREM arranged as direct repeatswith a 3 base spacer (DR3), direct repeats with a 6 base spacer (DR6) oreverted repeats with a 6 base spacer (ER6) (Table 1). In order toevaluate the affinity of these putative response elements for hPXR-RXRheterodimers, electromobility shift assays (EMSA) were performed usingwith in-vitro transcribed/translated hPXR and RXR. These showed thatXREM-DR3-1 and Prox-ER6 efficiently bind hPXR-RXR heterodimers resultingin a gel-shift, but no shift was apparent with XREM-DR6 and XREM-DR3-2.Despite this result, the present inventors have shown by site-directedmutagenesis experiments that the latter two response are functionallyimportant (FIG. 5). The XREM-ER6 did bind hPXR-RXR heterodimers, butwith less affinity than the XREM-DR3-1 or Prox-ER6.

TABLE 1 Sequences of putative nuclear receptor response elements withinthe CYP3A4 XREM compared to a putative response element in the proximalCYP3A4 5′-flanking region (Prox-ER6). An element from the proximal5′-flanking region of the rat CYP3A23 gene (CYP3A23 DR3) that interactswith the pregnane-X receptor is shown for comparitive purposes. SEQDesignation Sequence (core motifs in bold) ID NO: XREM-DR3-1 gaa TGAACTtgc TGACCC tct 2 XREM-ER6 cct TGAAAT catgtc GGTTCA agc 3 XREM-DR6 aggTGAATC acaagc TGAACT tct 4 XREM-DR3-2 ata TATTGT tat TGAACT atc 5Prox-ER6 ata TGAACT caaagg AGGTCA gtg 6 CYP3A23 DR3 aga TGAACT tcaTGAACT gtc 7

When the XREM is deleted from reporter gene constructs containing theCYP3A4 5′-flanking region, no xenobiotic-inducible activity is seen,demonstrating that the XREM region is essential for the process ofxenobiotic induction. Moreover, when the XREM is linked to aheterologous gene promoter, such as a minimal herpes simplex virusthymidine kinase promoter, xenobiotic induction is observed,demonstrating that the XREM is not absolutely reliant on other elementswithin the CYP3A4 gene. However, for maximal xenobiotic responsiveness,the present inventors have determined that an ER6 element within theproximal promoter region of the CYP3A4 gene (Prox-ER6, Table 1) isrequired. It is important to appreciate that the Prox-ER6 element has noxenobiotic-inducible activity in the absence of the XREM.

From the above findings, an optimised DNA sequence for the analysis ofthe xenobiotic induction of the CYP3A4 gene has been determined. Itincludes the XREM region as well as bases −356 to +53 of the proximalCYP3A4 promoter and is termed construct #5 (FIG. 6). Typically, this DNAsequence is linked to a reporter gene and studied in a suitable cell orcell-free system. The present inventors have determined that the HepG2cell line cultured in Dulbecco's Modified Eagle's Medium (DMEM) with 10%foetal bovine serum, transfected using a commercially available reagent(FuGene-6, Boehringer Mannheim, Mannheim, Germany) performs well.

Tissue-Specific and Constitutive Expression of CYP3A Subfamily Genes

The mechanisms determining the tissue-restricted and constitutiveexpression of CYP3A4 are probably similar, if not the same.Understanding these aspects of gene regulation are critical to adefinitive explanation of why humans exhibit such markedinter-individual variability in CYP3A4-mediated drug metabolism.

Human CYP3A subfamily P450s are predominantly expressed in the liver.However, there is significant tissue-restricted extrahepatic expression.CYP3A4 is expressed in significant amounts in small bowel, colon andpancreas, as well as in breast tissue. The relative levels ofconstitutive expression in the liver and intestine show littleconcordance, suggesting that different mechanisms operate in eachtissue. While CYP3A4 is constitutively expressed in all adult humanlivers, there is a 10-fold variability of CYP3A4 mRNA between liversamples. Variability in intestinal expression is even more marked,with >30-fold variation being reported.

In the case of hepatic CYP3A4 expression, using reporter constructscontaining the 5′-flanking region of the CYP3A4 gene, the presentinventors have demonstrated that liver-specific factors are preferablyrequired to support CYP3A4 transcription. When a liver-derived cell line(HepG2) was compared to a non-liver-derived line (NIH-3T3) for theirability to support transcriptional activation, either in the presence orabsence of hPXR, the non-liver-derived cell line was markedly inferior.Clearly, this suggests an optional role for liver-specific transcriptionfactors. However, the present inventors know that HepG2 cells, despitebeing a human liver-derived cell line, do not constitutively expressCYP3A4 in significant amounts. The present inventors have found that anunusual human orphan nuclear receptor, termed the human constitutiveandrostane receptor-β (hCAR-β) [5], when cotransfected into HepG2 cellswith a CYP3A4 5′-flanking region reporter construct, causes a 6- to10-fold increase in reporter activity. hCAR-β requires no ligand totransactivate regulatory DNA sequences and it's expression is almostentirely restricted to the liver. Experiments performed using deletionconstructs show that the response of the CYP3A4 gene to the hCAR-βreceptor is dependant on the XREM. Also, as observed for xenobioticinduction with the hPXR, hCAR-β-mediated constitutive expression wasobserved when the XREM was linked to the minimal thymidine kinasepromoter, though at a lower level than observed with the nativepromoter. Preliminary experiments suggest that there is cooperativitybetween the XREMand the Prox-ER6 element in hCAR-β-mediated constitutiveexpression. However, the native promoter alone shows no response tohCAR-β cotransfection.

Results

Cytochrome P450 3A4 (CYP3A4), the predominant P450 expressed in adulthuman liver, is subject to transcriptional induction by variety ofstructurally unrelated xenobiotic compounds, including the antibioticrifampicin. The present inventors have transfected a human liver-derivedcell line (HepG2) with various CYP3A4-luciferase reporter geneconstructs containing a nested set of 5′-deletions of the CYP3A4promoter. Rifampicin-inducible transcription of the reporter gene wasonly observed with the longest construct. Rifampicin treatment of cellstransfected with the −13000/+53-luciferase construct resulted in a 3- to5 fold increase in reporter gene activity. This construct was activatedin a dose-dependent manner by rifampicin with maximal induction at 5 μM.A further set of deletion clones were prepared and the responsive regionlocalised to bases −7800 to −7200, approximately. Polymerase chainreaction-generated deletion mutants suggest that rather than beingdependent on a short cis-acting element, the rifampicin-response isreliant upon the integrity of larger region encompassing several hundredbases. This region, in conjunction with a minimal CYP3A4 promoter (−362to +53), was capable of conferring rifampicin-responsiveness on thereporter gene. The induction was independent of the orientation of the−7800/−7200 fragment and its position relative to the proximal promoterof CYP3A4. Heterologous reporter gene constructs, containing the distalenhancer region of CYP3A4 ligated to the herpes simplex virus thymidinekinase promoter, were also capable of inducing luciferase expressionfollowing rifampicin treatment. Nucleotide sequence analysis of thisregion revealed a number of putative transcription factor binding sites.In summary, the present inventors have identified an enhancer region inthe CYP3A4 gene capable of mediating transcriptional activation byrifampicin.

Using a series of deletion constructs of the CYP3A4 gene 5′-flankingregion, the present inventors have discovered a DNA sequence, lyingapproximately 7.5 kilobases 5′ to the transcription initiation site ofthe human P450 CYP3A4 gene, that is responsible for the transcriptionalinduction of the CYP3A4 gene by xenobiotic inducers, includingtherapeutic drugs. The present inventors have called this element a‘xenobiotic-responsive enhancer module’ (XREM). This work has initiallyperformed with the potent CYP3A4 inducer, rifampicin, an antibioticcommonly used to treat tuberculosis. Preliminary studies with otherdrugs, however, suggest that the XREM may be capable of responding to abroad range of chemical compounds. The present inventors have used theXREM-containing DNA sequence from CYP3A4 to construct cell culturemodels that respond to xenobiotic inducers by increasing thetranscription of the CYP3A4 gene in a manner analogous to that occurringin humans.

The present inventors cotransfected a CYP3A4 5′-flanking regionconstruct, containing the XREM and linked to a luciferase reporter, intoHepG2 cells either with or without a hPXR expression construct(pSG-hPXR). The addition of the hPXR substantially increased thetranscription rate of the reporter construct, both constitutively(treated with vehicle alone) and following the addition of a xenobioticinducer (5 μM rifampicin). The observation that addition of the hPXRalone increases the transcription rate of the CYP3A4 XREM-containingreporter indicates that the hPXR is capable of some transactivation inthe absence of ligand or that an endogenous ligand for the hPXR existswithin the HepG2 cells.

The hPXR has been shown to bind to cis-acting DNA response elements(PXRE) as a heterodimer with the retinoid-x receptor (RXR). The PXREseems to generally consist of a repeat of the sequence TGAACT, either asdirect or everted/inverted repeats. The present inventors haveidentified a number of putative nuclear receptor response elements(PXREs) within the XREM shown in Table 1.

The present inventors have performed gel mobility shift experiments with³²P-labelled oligonucleotide probes to determine if hPXR-RXRheterodimers were capable of binding to the putative PXREs within theXREM.

To further examine the contribution of the putative PXREs within theXREM region to the transcriptional induction of CYP3A4 by rifampicin, aseries of luciferase reporter constructs containing deletions and/orsite-directed mutagenesis of the PXREs were created (FIG. 5). Deletionof the region containing the PXRE XREM-DR3-1 (−7834 to −7610 bp)resulted in almost complete abrogation of transcriptional activity. Thiswas not entirely due to the loss of function of XREM-DR3-1 as sitedirected mutagenesis of this PXRE resulted in only a 47% loss ofactivity. Mutation of XREM-DR6 also decreased transcription rate (to 52%of the wild-type) despite the apparent failure of this motif to bind ahPXR-RXR heterodimer in the gel-shift experiment. It is possible thatthis site binds other transcription factors or is a low-affinity sitefor hPXR-RXR.

It can be appreciated that the putative PXRE within the proximalpromoter region which consists of the Prox-ER6 motif has no function onits own. However, site-directed mutagenesis of the Prox-ER6 in aconstruct containing the complete XREM does decrease the transcriptionrate (to 41% of the wild-type) (FIG. 5) suggesting cooperativity betweenthe XREM and Prox-ER6.

The XREM Mediates the Transcriptional Induction of the CYP3A4 Gene toMany Xenobiotics, not only Rifampicin

Many drugs induce the transcription of the CYP3A4 gene. Because of itspotent induction properties, the present inventors used rifampicin toidentify and characterise the XREM. To determine if an XREM-containingreporter construct was capable of responding to other xenobiotics, HepG2cells transfected with the XREM were treated with a range of drugs (FIG.4). It was found that RU-486 (mifaprostone) and clotrimazole induced theXREM construct in a manner analogous to that that occurs in vivo. Thepresent inventors have also examined a wider range of drugs known toinduce CYP3A4 in vivo and found that the in vitro model developedclosely reflects in vivo experience.

Uses for the Invention

The invention has several areas of application, including but notrestricted to, therapeutic drug development. In some of these areas ofapplication, XREM containing DNA constructs are used to determine theeffects of compounds on CYP3A gene transcription. These constructstypically include reporter genes to allow for the convenient measurementof gene activity. Such reporter genes include chloramphenicol acetyltransferase, luciferase, alkaline phosphatase and green fluorescentprotein, though any detectable gene product, including messenger RNA(mRNA) could be used. Such gene constructs are introduced transiently orpermanently into suitable cultured cells, or as a transgene intransgenic animals. Additional DNA constructs can be cotransfected alongwith the XREM-containing constructs to provide suitable conditions toanswer particular questions regarding CYP3A gene regulation. Suchcotransfected constructs include expression plasmids for hPXR and hCAR-βwhich the present inventors have demonstrated to interact with the XREM.Present studies have also shown that there are many protein-DNAinteractions within the XREM so it is likely that other cotransfectedconstructs will prove useful for certain situations.

Another area of application is the use of the XREM sequence to examinehuman genomic DNA for the presence of polymorphisms relevant to CYP3A4gene expression. Such polymorphisms may be linked to several areas ofuse including the prediction of drug metabolising ability and diseaseassociations.

The use of Cell or Transgenic Animal Models to Screen ChemicalSubstances for the Ability to Induce the Formation of the CYP3A4 Enzyme

Induction of CYP3A subfamily enzymes by therapeutic drugs is generallyconsidered to be an undesirable effect. Such induction may increase theelimination of the drug itself or co-administered drugs. This generallyrenders drugs less effective. The ability to screen potential drugs forCYP3A induction during the early development phase is a useful in thatsuch drugs may be discarded in favour of ones that do possess thisaction. The information gained from such drug screening also allows thedevelopment of structure-function relationships, which indicatemolecules or parts of molecules that have the propensity to induceCYP3A4. Such knowledge allows the use of rational drug design tosynthesize new compounds that have more favourable characteristics. Thisprocess can be applied to existing drugs that undesirably induce CYP3As,such as those listed in Table 2.

TABLE 2 A non-exclusive list of therapeutic drugs known to induce theexpression of the CYP3A4 gene. Drug Carbamazapine Clotrimazole and otherimidazole anti-mycotics Lovastatin Phenytoin Phenobarbitone RifampacinRifabutin RU-486

In addition to the above, there are potential therapeutic uses for drugsspecifically designed to induce or inhibit CYP3A enzyme formation astheir primary action. Inducers of CYP3A4 can be used to accelerate themetabolism of xenobiotic toxins or endogenously produced substances thatare CYP3A4 substrates. Inhibitors of CYP3A4 could be used to overcomethe undesirable induction of CYP3A4 by therapeutic drugs, such as thoselisted in Table 2. It will be appreciated that there are many potentialuses of drugs specifically designed to modulate CYP3A enzyme formation,other than those listed here. The XREM sequence is useful in identifyingsuch drugs.

The XREM as a Tool for Pharmacogenetic Analysis/Disease Susceptibility

There are marked inter-individual differences in the capacity of humansto metabolise xenobiotic compounds (such as therapeutic drugs) andendogenous compounds (such as endogenously-secreted hormones). At leastsome of these differences can be related to polymorphisms in the genesencoding the enzymes or transport proteins that interact with thesexenobiotic or endogenously produced compounds.

Members of the cytochrome P450 gene superfamily are involved in themetabolism of a large range of lipophilic substrates. CYP3A subfamilyP450s (such as CYP3A4) are particularly involved in the metabolism oftherapeutic drugs and endogenously produced steroid hormones. It is wellrecognised that there are marked inter-individual differences in themetabolism of CYP3A substrates (up to 20-fold), however, to date, nopolymorphisms within the CYP3A4 protein coding region of the CYP3A4 genehave been found to account for these differences. This strongly suggeststhat it is the regulation of CYP3A gene expression that accounts forthese inter-individual differences.

The present inventors have demonstrated that the XREM region of CYP3A4is an important regulatory element for the transcriptional control ofthe expression of this gene. It follows that polymorphisms within theXREM could significantly effect gene transcription and the expression ofCYP3A4 protein, thus explaining, at least in part, the inter-individualdifferences in CYP3A-mediated metabolism.

Rebbeck and colleagues have recently described a polymorphism in theproximal 5′-flanking region of the CYP3A4 gene that correlates with moresevere forms of prostate cancer [6] and a reduced incidence of secondaryleukaemia following cancer chemotherapy [7]. It is presumed that thispolymorphism caused these changes due to effects on CYP3A4 expressionthough this has yet to be proven.

Thus, use of the XREM sequence and function provides a potentiallypowerful tool to search for CYP3A gene polymorphisms. Screening for suchpolymorphisms would be of considerable usefulness in determining theability of an individual to metabolise drugs (pharmacogenetic analysis)or determine disease susceptibility (eg., prostate cancer). The methodfor carrying out such screening typically involves the amplification ofthe genomic DNA region of interest (in this case the XREM) usingpolymerase chain reaction (PCR). The PCR product can then be examinedfor polymorphisms using one of several techniques such as restrictionfragment length polymorphisms (RFLP), sequencing of DNA orsingle-stranded conformational polymorphisms (SSCP) of DNA.

The Use of the XREM as an Inducible Enhancer for Gene Expression in theBroader Context of Molecular Biology, Transgenics and Directed GeneExpression

The concept of using a portion of a cytochrome P450 gene as a geneticswitch has been previously demonstrated using the CYP1A1 gene. As theXREM region of the CYP3A4 gene is capable of regulating transcription inresponse to xenobiotics as well as certain steroid hormones, it ispotentially useful as a genetic switch within the broad context ofmolecular biology and directed gene expression. An example is theactivation of a transgene in the liver of an animal via aPXR/XREM-dependant mechanism.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the invention as shown inthe specific embodiments without departing from the spirit or scope ofthe invention as broadly described. The present embodiments are,therefore, to be considered in all respects as illustrative and notrestrictive.

REFERENCES

-   1. Lehmann J M, et al. (1998) The human orphan nuclear receptor PXR    is activated by compounds that regulate CYP3A4 gene expression and    cause drug interactions J Clin invest 102:1016–1023.-   2. Bertilsson G, at al. (1998) Identification of a human nuclear    receptor defines a new pathway for CYP3A induction. Proc Natl Acad    Sci USA 95:12208–12213.-   3. Blumberg B, et al. (1998) SXR, a novel steroid and xenobiotic    sensing receptor. Genes & Dev 12:3195–3205.-   4. Mangelsdorf D J, et al. (1995) The nuclear receptor superfamily:    the second decade. Cell 83:835–839.-   5. Baes M, et al. (1994) A new orphan member of the nuclear hormone    receptor superfamily that interacts with a subset of retinoic acid    response elements. Mol Cell Biol 14:1544–52.-   6. Rebbeck T R, et al. (1998) Modification of the clinical    presentation of prostate tumors by a novel genetic variant in    CYP3A4. J Natl Cancer Inst 90:1225–1229.-   7. Felix C A, et al. (1998) Association of CYP3A4 genotype with    treatment-related leukemia. Proc Natl Acad Sci USA 95:13176–81.

1. A substantially purified nucleic acid molecule comprising thesequence SEQ ID NO:
 1. 2. A vector comprising the substantially purifiednucleic acid molecule according to claim
 1. 3. The vector according toclaim 2 further comprising a gene for encoding a reporter molecule. 4.The vector according to claim 3 wherein the reporter molecule is fireflyluciferase, beta-galactosidase, alkaline phosphatase, green fluorescentprotein or chloramphenicol acetyl transferase.
 5. A cell comprising thevector according to claim
 2. 6. The cell according to claim 5 whereinthe cell is a hepatic cell.